Substances that inhibit vascular neogenesis are expected to be applied as antitumor agents since they inhibit cancer growth, invasion and metastasis, and angiostatin is known to be one of such substances. Angiostatin is a protein having a molecular weight of about 40,000 that is obtained by the degradation of a fibrinolytic factor, plasminogen, present in blood. It has been reported to demonstrate dramatic effects against cancer in animal studies (see Non-Patent Document 1). There are two methods used to produce angiostatin, namely (1) hydrolyzing plasminogen with a protease known as elastase (see Non-Patent Document 2), and (2) producing it directly in Escherichia coli using gene recombination technology (see Non-Patent Document 3). In the case of method (1), there is considerable formation of by-products due to the low substrate-specificity of elastase, thus making it difficult to selectively form angiostatin from plasminogen. In addition, this method also has the disadvantage of the activity of the resulting angiostatin being low. In the case of method (2), it is difficult to purify angiostatin produced from E. Coli, and considerable costs are incurred. In addition, there is also a problem with the low level of solubility. Consequently, there has been a need to develop a means of obtaining angiostatin at high purity using a simple method.
Recently, a protease produced by Bacillus megaterium strain A9542 known as bacillolysin MA has been observed to form an angiostatin-like fragment having angiogenesis inhibitory action by specifically severing plasminogen (comprising mainly of Glu1-Ser441, hereinafter referred to as BL-angiostatin), and a mini-plasminogen-like fragment having thrombolytic activity (comprising mainly of Val442-Asn791) (see Patent Document 1). Since this enzyme known as bacillolysin MA is extremely stable, it can be used in various applications by immobilizing on a support. Therefore, there is a need to develop a process for obtaining highly pure BL-angiostatin by carrying out the steps from the reaction of bacillolysin MA and substrate plasminogen to purification of the resulting product BL-angiostatin in a single stage using the characteristics of bacillolysin MA, and to develop a device for carrying out that process.
On the other hand, enzyme-immobilized reactors in which various enzymes such as protease which can be used for various uses are immobilized on a support for use in a reaction have been proposed in the prior art. However, proteases are susceptible to inactivation resulting from degradation of the immobilized protease due to self-digestion, and in the case the substrate of the enzyme such as protease has a high molecular weight, the reaction between the enzyme bound to the support and the substrate has difficulty in proceeding efficiently and rapidly due to spatial restrictions. For these reasons, the applications of enzyme-immobilized reactors are limited by the types of enzymes and substrates used, and expectations have been placed on the development of an enzyme-immobilized reactor that can be widely used for various types of enzymes and substrates.
Patent Document 1: Japanese Unexamined Patent Publication No. 2002-272453
Non-Patent Document 1: M. S. O'Reilly, et al., Cell (USA), Oct. 21, 1994, Vol. 79, No. 2, pp. 315-328
Non-Patent Document 2: M. S. O'Reilly, et al., Nature Medicine (USA), 1996, Vol. 2, pp. 689-692
Non-Patent Document 3: B. K. Sim, et al., Cancer Research (USA), 1997, Vol. 57, pp. 1329-1334
As a result of conducting extensive research to develop an enzyme-immobilized reactor free of the aforementioned problems, as well as a process of producing BL-angiostatin using the characteristics of the aforementioned bacillolysin MA, the inventor of the present invention found that, by binding an enzyme such as bacillolysin MA together with a molecule that specifically binds with an enzyme substrate to a support to compose an affinity trap reactor, together with obtaining a reactor that is free of the aforementioned problems, by using an affinity trap reactor in which bacillolysin MA is immobilized as the enzyme and Lys is immobilized as the molecule that specifically binds with the enzyme substrate, a process is obtained for highly efficiently and rapidly degrading and purifying BL-angiostatin from plasminogen contained in biological samples such as blood, thereby leading to completion of the present invention.